The invention relates to a method for producing L-cystine by fermentation.
L-cystine is a disulfide which is formed by the oxidation of two molecules of L-cysteine. This reaction is reversible, which means that L-cystine may be reconverted to L-cysteine by reduction.
The amino acid L-cysteine is of economic significance. It is used, for example, as a food additive (particularly in the baking industry), as a feedstock in cosmetics, and also as a starting product for preparing active pharmaceutical ingredients (particularly N-acetylcysteine and S-carboxymethylcysteine).
L-cysteine plays a key role in sulfur metabolism in all organisms and is used in the synthesis of proteins, glutathione, biotin, lipoic acid, methionine and other sulfur-containing metabolites. In addition, L-cysteine serves as precursor for the biosynthesis of coenzyme A. The biosynthesis of L-cysteine has been investigated in depth in bacteria, particularly in Enterobacteria, and is described in detail in Kredich (1996, Biosynthesis of cysteine, pp. 514-527. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C.).
In addition to the classical production of L-cysteine by means of extraction from keratin-containing material such as hair, bristles, horns, hooves and feathers or by means of biotransformation by enzymatic conversion of precursors, a method for producing L-cysteine by fermentation was also developed some years ago. The prior art with respect to the production of L-cysteine by fermentation using microorganisms has been described in detail e.g. in U.S. Pat. Nos. 6,218,168B1, 5,972,663A, U.S.2004/0038352A1, CA2386539A1, U.S.2009/0053778A1 and U.S.2009/0226984A1. The bacterial host organisms used here are, inter alia, strains of the genus Corynebacterium and also representatives from the Enterobacteriaceae family such as Escherichia coli or Pantoea ananatis. 
In addition to the classical procedure of attaining improved L-cysteine producers by mutation and selection, specific genetic modifications to the strains have also been carried out in order to achieve an effective L-cysteine overproduction.
The insertion of a cysE allele coding for a serine O-acetyl transferase having a reduced feedback inhibition by L-cysteine thus led to an increase in cysteine production (U.S. Pat. No. 6,218,168B1; Nakamori et al., 1998, Appl. Env. Microbiol. 64: 1607-1611; Takagi et al., 1999, FEBS Lett. 452: 323-327). By means of a feedback-resistant CysE enzyme, the formation of O-acetyl-L-serine, the direct precursor of L-cysteine, is largely decoupled from the L-cysteine level in the cell.
O-acetyl-L-serine is formed from L-serine and acetyl-CoA. Thus, the provision of L-serine in sufficient amounts for L-cysteine production is of major importance. This may be achieved by insertion of a serA allele coding for a 3-phosphoglycerate dehydrogenase having a reduced ability for feedback inhibition by L-serine. The formation of 3-hydroxypyruvate, a precursor of L-serine, is thereby largely decoupled from the L-serine level in the cell. Examples of such SerA enzymes have been described in EP0620853, U.S. 2005/0009162 A1, U.S.2005009162A2 and EP0931833.
It is further known that the L-cysteine yield in the fermentation may be increased by weakening or destroying genes coding for L-cysteine-degrading enzymes, such as the tryptophanase TnaA or the cystathionine-(β-lyases MalY or MetC (EP1571223).
The increase of the transport of L-cysteine out of the cell is a further possibility to increase the product yield in the medium. This may be achieved by overexpression of so-called efflux genes. These genes code for membrane-bound proteins which mediate the export of L-cysteine from the cell. Various efflux genes for L-cysteine export have been described (U.S. Pat. No. 5,972,663A, U.S.2004/0038352A1, U.S.2005221453, WO2004113373).
The export of L-cysteine from the cell into the fermentation medium has several advantages:    1) L-cysteine is continuously abstracted from the intracellular reaction equilibrium with the result that this amino acid is maintained at a low level in the cell and there is therefore no feedback inhibition of sensitive enzymes by L-cysteine:L-cysteine (intracellular)⇄L-cysteine (medium)  (1)    2) The L-cysteine released into the medium is oxidized to the disulfide L-cystine in the presence of oxygen, which is introduced into the medium during the cultivation (U.S. Pat. No. 5,972,663A):2 L-cysteine+½O2⇄L-cystine+H2O   (2)
Since the solubility of L-cystine in aqueous solution at a neutral pH is only very low, especially in comparison to L-cysteine, the disulfide precipitates even at a low concentration and forms a white precipitate:L-cystine (dissolved)⇄L-cystine (precipitate)  (3)
Through the precipitation of L-cystine, the level of the product dissolved in the medium is reduced, whereby the reaction equilibrium of (1) and (2) in each case is also drawn to the product side.    3) The technical effort for the purification of the product is considerably lower if the amino acid can be obtained directly from the fermentation medium than when the product accumulates intracellularly and an initial cell lysis has to be carried out.
Facultative anaerobic bacteria, such as E. coli or P. ananatis, grow better under aerobic conditions, i.e. in the presence of oxygen, since they can obtain more energy from carbohydrates such as glucose by aerobic metabolism than by fermentation (anaerobic metabolism) of the respective energy source.
L-cysteine production using E. coli or P. ananatis is also carried out under aerobic conditions (U.S.2004/0038352A1, U.S. Pat. No. 5,972,663A, CA2386539A1, EP1389427, EP2138585). It is disclosed in the methods described in more detail that the oxygen input into the culture is adjusted such that the culture broth has an oxygen content of 50% during the fermentation.
A disadvantage of the methods described for the production of L-cysteine by fermentation is that the amino acid is present in the culture broth in various forms. In addition to the precipitated L-cystine in the precipitate, L-cystine in dissolved form and also L-cysteine and also a thiazolidine are found in the culture supernatant (U.S. Pat. Nos. 6,218,168B1, 5,972,663A, CA2386539A1). This thiazolidine (2-methylthiazolidine-2,4-dicarboxylic acid) is the condensation product of L-cysteine and pyruvate, which is formed in a purely chemical reaction.
The term “total cysteine” in the context of this invention combines L-cysteine and the L-cystine and thiazolidine compounds formed therefrom, which are formed during the fermentation and accumulate in the culture supernatant and in the precipitate.
In the known methods, the proportion of precipitated L-cystine varies at the end of the fermentation. This is between 40-66% (U.S. Pat. No. 5,972,663A, CA2386539A1) of the total cysteine, which means that the residual 34-60% of the total cysteine is present in the culture supernatant, namely predominantly in the form of L-cysteine and thiazolidine. This product heterogeneity hinders the recovery and purification of the product from the culture broth.
A method is therefore desirable in which the end product mostly occurs in only one form. Particularly favorable is a method in which L-cystine is predominantly formed as product, since L-cystine precipitates even at a low concentration due to its low solubility in a pH neutral aqueous medium, whereby the reaction equilibrium is shifted to the product side, which in turn leads to relatively high product yields.